Gastric cancer has been epidemiologically linked to infection with the bacterium Helicobacter pylori (HP). Mechanisms underlying this possible connection are unknown. In HP infection, chronic gastritis occurs, characterized by the activation of mucosal macrophages and other immune cells. In this inflamed tissue, resident and recruited immune cells may be activated directly by bacterial products or indirectly by intracellular signaling. With stimulation, nitric oxide (NO) can be abundantly produced by the inducible form of the enzyme NO synthase (iNOS) in many cell types, especially immune cells. Our preliminary data indicate that HP dramatically increases iNOS mRNA and NO production in macrophages. NO has been shown to possess mutagenic activity. We therefore hypothesize that one mechanism by which HP contributes to the development of gastric adenocarcinoma is the chronic upregulation of NO production in gastric mucosa. Our proposal will have two main Specific Aims: I) To discover mechanisms of HP-induced NO production in macrophages, and II) To demonstrate that HP-induced NO production contributes to gastric carcinogenesis. In order to accomplish Aim I, we will: A) prove that functional iNOS is directly induced by HP in macrophages by measuring I) production of nitrite, a stable metabolite of NO, 2) iNOS enzyme activity, 3) iNOS mRNA expression by Northern blotting, and 4) iNOS protein expression by Western blotting; B) identify and purify HP-derived factors mediating iNOS induction in macrophages; C) determine whether luminal HP stimulates gastric epithelial-to-macrophage signaling using 1) a co- culture model with HP-treated epithelial cells grown on semipermeable membrane supports above macrophages, and 2) HP-free conditioned media from HP-stimulated and control epithelial cells; and D) study the mechanism of HP-induced activation of iNOS in macrophages, specifically mechanisms of transcriptional regulation of iNOS. For Aim II, we will: A) demonstrate mutagenic effects of NO on gastric epithelial cells, specifically microsatellite instability and mutations in the gastric cancer suppressor genes p53 and APC; B) correlate gastric mucosal iNOS gene expression with HP status in gastric biopsies of patients; and C) correlate in vivo HP status and iNOS expression with gastric carcinogenesis. To accomplish this last sub-Aim, we will: l) correlate HP serology with malignant tissue mutational events in gastric cancer patients from countries with relatively high (S. Korea) and low (U.S.A.) HP rates; 2) ascertain HP serology and tissue HP status, iNOS expression, and mutational events at discrete stages of gastric carcinogenesis, including active superficial gastritis, chronic atrophic gastritis, intestinal metaplasia of the stomach, gastric dysplasia, and frank adenocarcinoma. If iNOS induction participates in the initiation or progression of gastric cancer, an understanding of the factors mediating induction of iNOS by HP should provide insight into possible novel targets of pharmacologic intervention or prevention in gastric carcinogenesis as well as potential novel diagnostic markers of gastric neoplastic progression.